Differential cytokine expression in avian cells in response to invasion ...

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encoded on Salmonella pathogenicity island 1 (Darwin .... 1–2 week old Rhode Island Red chicks as previously described ...... PhD thesis, University of Bristol.
Microbiology (2000), 146, 3217–3226

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Differential cytokine expression in avian cells in response to invasion by Salmonella typhimurium, Salmonella enteritidis and Salmonella gallinarum Pete Kaiser1, Lisa Rothwell1, Edouard E. Galyov1, Paul A. Barrow1, Joan Burnside2 and Paul Wigley1† Author for correspondence : Paul Wigley. Tel : j44 116 255 1551. Fax : j44 116 257 7631. e-mail : Microwig!aol.com

1

Institute for Animal Health, Compton, Berkshire RG20 7NN, UK

2

Department of Animal and Food Sciences, University of Delaware, 531 South College Avenue, Newark, DE 19717-1303, USA

Salmonella enterica is a facultative intracellular pathogen that is capable of causing disease in a range of hosts. Although human salmonellosis is frequently associated with consumption of contaminated poultry and eggs, and the serotypes Salmonella gallinarum and Salmonella pullorum are important world-wide pathogens of poultry, little is understood of the mechanisms of pathogenesis of Salmonella in the chicken. Type III secretion systems play a key role in host cell invasiveness and trigger the production of pro-inflammatory cytokines during invasion of mammalian hosts. This results in a polymorphonuclear cell influx that contributes to the resulting enteritis. In this study, a chicken primary cell culture model was used to investigate the cytokine responses to entry by the broad host range serotypes S. enteritidis and S. typhimurium, and the host specific serotype S. gallinarum, which rarely causes disease outside its main host, the chicken. The cytokines interleukin (IL)1β, IL-2, IL-6 and interferon (IFN)-γ were measured by quantitative RT-PCR, and production of IL-6 and IFN-γ was also determined through bioassays. All serotypes were invasive and had little effect on the production of IFN-γ compared with non-infected cells ; S. enteritidis invasion caused a slight downregulation of IL-2 production. For IL-1β production, infection with S. typhimurium had little effect, whilst infection with S. gallinarum or S. enteritidis caused a reduction in IL-1β mRNA levels. Invasion of S. typhimurium and S. enteritidis caused an eight- to tenfold increase in production of the proinflammatory cytokine IL-6, whilst invasion by S. gallinarum caused no increase. These findings correlate with the pathogenesis of Salmonella in poultry. S. typhimurium and S. enteritidis invasion produces a strong inflammatory response, that may limit the spread of Salmonella largely to the gut, whilst S. gallinarum does not induce an inflammatory response and may not be limited by the immune system, leading to the severe systemic disease fowl typhoid.

Keywords : Salmonella, cytokines, interleukin-6, chicken, inflammatory response

INTRODUCTION

Salmonella enterica is a facultative intracellular patho-

gen capable of causing disease in a wide range of host species. S. enterica serotypes may be divided into two groups on the basis of disease caused and their host

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† Present address : School of Pharmacy and Pharmaceutical Sciences, DeMontfort University, Leicester LE1 9BH, UK. Abbreviations : CKC, chick kidney cells ; CM, conditioned medium ; Ct, threshold cycle value ; DMEM, Dulbecco’s modified Eagle’s medium ; FAM, 5carboxyfluorescein ; FBS, foetal bovine serum ; IFN, interferon ; IL, interleukin ; PMN, polymorphonuclear cell ; P/S, penicillin and streptomycin ; rm, recombinant murine ; TAMRA, N,N,N,Nh-tetramethyl-6-carboxyrhodamine. The GenBank accession numbers for the sequences reported in this paper are AI982185 for chicken IL-6 cDNA and AJ250838 for the partial chicken IL-6 genomic sequence, respectively. 0002-4081 # 2000 SGM

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P. K A I S E R a n d O T H E R S

species range (Barrow, 1996). Broad host range serotypes including Salmonella typhimurium and Salmonella enteritidis can cause enteritis in a wide range of host species, these two serotypes being responsible for the majority of Salmonella food-borne enteritis in man. Around 30 000 cases of human salmonellosis are reported in the United Kingdom each year (Wall & Ward, 1999) and are often associated with consumption of contaminated poultry meat or eggs (Humphrey, 1999). However, with the exception of very young chicks, S. enteritidis and S. typhimurium rarely cause clinical disease, but can colonize the gut of poultry (Barrow et al., 1987 ; Barrow & Lovell, 1990). Salmonellae may then be shed in the faeces and can lead to horizontal transmission to other birds in the flock by the faecal–oral route and to contamination of meat by faeces at the time of slaughter. S. enteritidis may also colonize the reproductive tract, leading to the contamination of eggs (reviewed by Humphrey, 1999). Restricted host range serotypes rarely cause disease outside their natural host, in which they cause systemic typhoid-like disease. Examples of these serotypes include Salmonella typhi in man, and Salmonella pullorum and Salmonella gallinarum in the chicken, which cause pullorum disease and fowl typhoid respectively (Snoeyenbos, 1991 ; Pomeroy & Nagaraja, 1991). These diseases have been largely controlled in Europe and North America, but still cause substantial losses of poultry in South America and Asia where intensification of the poultry industry is in its infancy. Recent reports suggest that differences in the disease caused by ‘ typhoid-like ’ restricted host range serotypes and enteritis caused by broad host range serotypes may be the results of differences in the early stages of pathogenesis (Weinstein et al., 1998 ; Henderson et al., 1999). Following oral infection by S. typhimurium in mammals, Salmonella penetrates through the intestinal epithelium (Gala! n & Sansonetti, 1996). Entry into epithelial cells is mediated by a type III secretion system encoded on Salmonella pathogenicity island 1 (Darwin & Miller, 1999). The proteins secreted by this system interact with the epithelial cells, triggering a number of responses including the production of pro-inflammatory cytokines (Gala! n & Sansonetti, 1996 ; Darwin & Miller, 1999), leading to an influx of polymorphonuclear cells (PMNs) accompanied by increased fluid secretion. The interaction of Salmonella with epithelial cells results in a number of responses including damage to the intestinal epithelium and diarrhoea. S. typhimurium is also capable of causing gastroenteritis in young chicks and causes intestinal lesions, but not clinical disease, in older birds (Barrow et al., 1987). Relatively little is known about the molecular mechanisms of Salmonella entry in the chicken gut, though the basic mechanisms of pathogenesis of S. typhimurium appear to be similar to those in mammals (Henderson et al., 1999). Invasion causes rapid inflammation of the intestinal mucosa and infiltration of large numbers of heterophils, the avian equivalent of neutrophils, followed by macrophages, resulting in intestinal lesions. In contrast, following 3218

infection with S. pullorum, rapid inflammation does not occur, and only small numbers of heterophils are found associated with the intestinal epithelium (Henderson et al., 1999). S. pullorum is less invasive than S. typhimurium in a range of avian and mammalian primary and continuous cell lines. S. gallinarum has also previously been shown to be less invasive than S. typhimurium in both avian and mammalian cells (Barrow & Lovell, 1989). Greater understanding of how Salmonella interacts with the host is needed to understand the disease and colonization processes in the chicken both in terms of animal and public health. Recent progress in the cloning of avian cytokines has led to the development of reagents with which to measure cytokine production in response to infection in the chicken, and this may allow a greater insight into interactions between the host and salmonellae at a cellular and molecular level. The avian orthologues of the Th1 cytokines interferon-γ (IFN-γ) and interleukin-2 (IL-2) have recently been cloned (Digby & Lowenthal, 1995 ; Sundick & Gill-Dixon, 1997), as have the pro-inflammatory cytokines IL-1β (Weining et al., 1998) and IL-6 (accession no. AI982185). Reproducible, sensitive bioassays exist to measure chicken IFN-γ-like (Lowenthal et al., 1995) and IL-6-like (Lynagh, 1998 ; Nakamura et al., 1998) activities. The genomic sequences and gene structure for IFN-γ (Kaiser et al., 1998), IL-2 (Kaiser & Mariani, 1999) and IL-1β (accession no. AJ245728) have been fully determined. A partial genomic sequence for IL-6 has also recently been isolated (accession no. AJ250838). Gene structure information makes possible the design of probes and primers to specifically quantify cytokine mRNA levels using real-time quantitative RT-PCR. We aimed to determine the levels of cytokines, particularly the pro-inflammatory cytokines IL-6 and IL-1β, produced following the invasion of the broad host range serotypes S. typhimurium and S. enteritidis, and the host specific serotype S. gallinarum, into chicken cells in vitro. Primary chick kidney cells (CKC) were chosen as the host cell for the invasion model ; there is no current suitable chicken intestinal epithelium model. CKC contain a high proportion of epithelial cells, few phagocytic cells and have previously been shown to be invaded by a range of Salmonella serotypes (Barrow & Lovell, 1989). Different levels of cytokine production following invasion with the broad host range serotypes (S. typhimurium and S. enteritidis), compared to the host specific serotype (S. gallinarum), would provide insight into the mechanisms of immunopathogenesis of salmonellosis in the chicken. METHODS Bacterial strains. The following well-characterized strains were selected as representative of their serotype for their in vitro behaviour in the chicken : Salmonella gallinarum 9 causes severe systemic disease in adults and chicks (Smith, 1955), Salmonella typhimurium Phage Type (PT)14 strain F98

Salmonella and cytokine expression Table 1. Real-time quantitative RT-PCR probes and primers RNA target 28S

IFN-γ

IL-1β

IL-2

IL-6

Probe/primer sequence (5h–3h)

Probe F R Probe F R Probe F R Probe F R Probe F R

(VIC)-AGGACCGCTACGGACCTCCACCA-(TAMRA) GGCGAAGCCAGAGGAAACT GACGACCGATTTGCACGTC (FAM)-TGGCCAAGCTCCCGATGAACGA-(TAMRA) GTGAAGAAGGTGAAAGATATCATGGA GCTTTGCGCTGGATTCTCA (FAM)-CCACACTGCAGCTGGAGGAAGCC-(TAMRA) GCTCTACATGTCGTGTGTGATGAG TGTCGATGTCCCGCATGA (FAM)-ACTGAGACCCAGGAGTGCACCCAGC-(TAMRA) TTGGAAAATATCAAGAACAAGATTCATC TCCCAGGTAACACTGCAGAGTTT (FAM)-AGGAGAAATGCCTGACGAAGCTCTCCA-(TAMRA) GCTCGCCGGCTTCGA GGTAGGTCTGAAAGGCGAACAG

Exon boundary

Accession no.* X59733

3\4

Y07922

5\6

AJ245728

2\3

AJ009800

3\4

AJ250838

* Refers to the genomic DNA sequence.

(Smith & Tucker, 1975) and Salmonella enteritidis PT4 strain 125589 (Barrow & Lovell, 1990) cause disease in young chicks but colonize older birds without disease. Salmonella dublin 2229 (Baird et al., 1985) and Escherichia coli K-12 (Smith, 1978) were also tested. With the exception of S. dublin 2229, the invasiveness of all strains in primary chicken cell lines has been previously determined (Barrow & Lovell, 1989). Bacteria were cultured in Luria–Bertani (LB) broth (Difco) at 37 mC in an orbital shaking incubator at 150 r.p.m. Cell culture. Primary CKC were prepared from the kidneys of 1–2 week old Rhode Island Red chicks as previously described (Barrow & Lovell, 1989). Briefly, kidneys were removed aseptically, teased apart and trypsinized with versene (0n8 % NaCl, 0n02 % KH PO , 0n15 % Na HPO , 0n02 % KCl and # % 0n02 % EDTA). #Cell% concentrations were adjusted to 1i10' ml−" in complete DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 12n5 % (v\v) heat-inactivated foetal bovine serum (FBS), 10 % (v\v) tryptose phosphate broth (Difco), 25 U nystatin ml−", 100 U penicillin ml−" and 1 µg streptomycin ml−" (P\S), at pH 7n0 and grown in 1 ml per well in 24-well Nunclon plates (Nunc) for 72 h at 37 mC, 5 % CO . Two hours prior to use in invasion assays, the media was # replaced with DMEM without antibiotics. HD11 (a chicken macrophage cell line) cells (Beug et al., 1979) were seeded at 4i10& ml−" and grown at 41 mC, 5 % CO in # RPMI 1640 medium (Life Technologies) containing 20 mM -glutamine (Life Technologies), 2n5 % FBS, 2n5 % chicken serum, 10 % tryptose phosphate broth and P\S. 7TD1 (an IL6-dependent mouse plasmacytoma cell line) cells (van Snick et al., 1986) were seeded at 2i10& ml−" and grown at 37 mC, 5 % CO in RPMI 1640 medium containing 20 mM -glutamine, # FBS, 0n05 mM 2-mercaptoethanol and P\S. During 10 % routine culture, recombinant murine (rm) IL-6 was added at 10 pg ml−". Invasion of cells. Bacterial cultures were diluted in LB to 1i10) ml−" and 100 µl added to the CKC to give a m.o.i. of 10 bacteria per chicken cell. Cells were incubated for either 2 or 4 h at 37 mC, 5 % CO . After incubation, cell supernatants were removed, filtered #through a 0n22 µm filter and stored at k20 mC prior to determination of cytokine production.

Extracellular bacteria were then killed by incubating cells for 1 h at 37 mC in DMEM containing 100 µg gentamicin ml−" (Sigma). Cells were washed three times in Hank’s buffered saline solution (Life Technologies) and lysed with 1 ml 1 % (v\v) Triton X-100 (Sigma) for 30 min at 37 mC. Viable counts of the intracellular bacteria in the lysate were made on LB agar. Each assay was performed in triplicate for each serotype. Statistical analysis of variance between species and serotypes was made using the Minitab for Windows v12.21 statistical program (Minitab). The probability level for significance was taken as P 0n05. Real-time quantitative RT-PCR. Cytokine mRNA levels in

infected and control CKC cultures were quantified using a method based on that of Moody et al. (2000). Total RNA was prepared from CKC cultures using the RNeasy mini kit (Qiagen) following the manufacturer’s instructions. Purified RNA was eluted in 50 µl RNase-free water and stored at k70 mC. For both cytokine and 28S rRNA-specific amplification, primers and probes were designed using the Primer Express software program (PE Applied Biosystems). Details of the probes and primers are given in Table 1. All cytokine probes were designed, from the sequence of the relevant genes, to lie across intron–exon boundaries. Cytokine probes were labelled with the fluorescent reporter dye 5-carboxyfluorescein (FAM) at the 5h end and the quencher N,N,N,Nh-tetramethyl-6carboxyrhodamine (TAMRA) at the 3h end. The 28S probe was labelled with the fluorescent reporter dye VIC (PE Applied Biosystems) at the 5h end and TAMRA at the 3h end. RT-PCR was performed using the TaqMan EZ RT-PCR kit (PE Applied Biosystems). The RT-PCR mixture consisted of 1i EZ RT-PCR buffer [including 60 nM 6-carboxy-x-rhodamine (a fluorescent reference dye)], 3 mM manganese acetate, 300 µM dATP, dCTP and dGTP, 600 µM dUTP, 600 nM each primer, 100 nM probe, 0n1 U rTth polymerase, 0n01 U AmpErase UNG (uracil-N-glycosylase), 5 µl total RNA, made up to 25 µl with water. Amplification and detection of specific products were performed using the ABI PRISM 7700 Sequence Detection System (PE Applied Bio3219

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Table 2. Standard curve data from real-time quantitative RT-PCRs on total RNA extracted from stimulated splenocytes Target

∆Rn*

significance threshold 28S IFN-γ IL-1β IL-2 IL-6

0n05 0n01 0n02 0n01 0n02

Log dilutions

Ct values†

R2‡

Slope

10−"–10−& 10−"–10−& 10−"–10−& 10−"–10−% 10−"–10−%

8–22 16–31 22–35 24–34 23–36

0n9719 0n9933 0n9903 0n9980 0n9832

3n138 3n503 3n124 3n122 4n333

* ∆Rn l change in the reporter dye. † Ct l threshold cycle value, the cycle at which the change in the reporter dye levels detected passes the ∆Rn. ‡ R# l coefficient of regression.

systems) with the following cycle profile : 1 cycle of 50 mC for 2 min, 96 mC for 5 min, 60 mC for 30 min and 95 mC for 5 min, and 40 cycles of 94 mC for 20 s, 59 mC for 1 min. Quantification was based on the increased fluorescence detected by the ABI PRISM 7700 Sequence Detection System due to hydrolysis of the target-specific probes by the 5h nuclease activity of the rTth DNA polymerase during PCR amplification. The passive reference dye 6-carboxy-x-rhodamine, which is not involved in amplification, was used to correct for fluorescent fluctuations resulting from changes in the reaction conditions, for normalization of the reporter signal. Results are expressed in terms of the threshold cycle value (Ct), the cycle at which the change in the reporter dye (∆Rn) passes a significance threshold. In this work, the threshold values of ∆Rn are as shown in Table 2, for all reactions described. To generate standard curves for the cytokine and 28S rRNAspecific reactions, total RNA extracted from stimulated splenocytes was serially diluted in sterile RNase-free water and dilutions made from 10−" to 10−&. Each RT-PCR experiment contained three no-template controls, test samples and a log dilution series. Each experiment was performed in triplicate,"!with replicates performed on different days. Regression analysis of the mean values of six replicate RT-PCRs for the log diluted RNA was used to generate standard "! curves.

measured in a Titretek Multiscan MCC\340 ELISA reader (ICN) at 543 nm. Serial dilutions of sodium nitrite (Sigma) were used to determine a standard curve. Data are expressed as µM NO− (5i10% cells)−" (48 h)−". IFN-γ-like activity of the # was determined using log-linear regression test samples analysis against the NO− standard curve. # To confirm the specificity of the NO−-inducing activity, the # to their addition to CM were also pre-incubated for 1 h, prior the HD11 cells, with either 1E12, an anti-chicken IFN-γ neutralizing mAb (Lambrecht et al., 2000), or the LPS inhibitor polymyxin B. 7TD1 bioassay. The 7TD1 bioassay was based on that described by van Snick et al. (1986). Cells were plated at 2i10$ cells per well in 96-well plates and then cultured at 37 mC, 5 % CO for 72 h in the presence of a variety of stimuli. The presence #of IL-6-like activity was indicated by proliferation, as measured by the incorporation of [$H]thymidine (0n5 µCi [$H]thymidine per well) during the final 6 h incubation. IL-6-like factor activity of the test samples was determined using log-linear regression analysis against rmIL6 standards. The dynamic range of the rmIL-6 standard was determined from the linear part of the curve and plotted on the same dilution scale as the samples. This range was then used to regress the curve of the test sample back to the level of half maximal proliferation seen in the rmIL-6 standard curve. The dilution of test sample required to bring about this level of proliferation was referred to as one dilution unit. Taking into account the dilution factor, the number of dilution units in each test sample was determined. The number of dilution units in samples run in separate assays was compared by regressing each sample using the standard curve run within the same assay.

RESULTS Invasion of CKC cells by Salmonella and E. coli

All four Salmonella serotypes invaded CKC at levels higher than the non-invasive E. coli control (P 0n05). S. typhimurium was significantly more invasive than S. gallinarum, S. enteritidis or S. dublin (P 0n001). No significant difference in invasion was found between S. gallinarum, S. enteritidis or S. dublin (P  0n2). Results for a 2 h contact time are shown in Table 3. Invasion

Table 3. Invasion of CKC with Salmonella serotypes and E. coli

Macrophage activation factor assay. Macrophage activation

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factor activity, typically used in the chicken as a measure of IFN-γ activity (Lowenthal et al., 1995), was assayed by the production of nitric oxide by stimulated HD11 cells (Beug et al., 1979), quantitated by the accumulation of nitrite (NO−) in # the culture medium (Ding et al., 1988 ; Sung et al., 1991). HD11 cells were seeded at a density of 5i10% cells per well in flat-bottomed 96-well microtitre plates (Corning) in growth medium (as described above). Subsequently, triplicate 100 µl samples of twofold serial dilutions of CKC conditioned medium (CM) were added and the cells cultured for 48 h at 41 mC, 5 % CO . The nitrite concentration was assayed by # mixing 100 µl cell-free culture supernatant with 100 µl Griess reagent [0n3 %, w\v, naphthylethylenediamine dihydrochloride (Sigma), 1 % (w\v) sulphanilamide (Sigma) in 2n5 % H PO (BDH)] and incubating for 10 min at room tem$ % perature. The absorbance of the reaction product was

Values are meansp of three repeats of the experiment. Bacteria had a contact time of 2 h with the CKC prior to killing of extracellular bacteria with 100 µg gentamicin ml−".

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Bacterial strain

S. typhimurium F98 S. gallinarum 9 S. dublin 2229 S. enteritidis 125589 E. coli K-12

Log10 c.f.u. invading bacteria (ml CKC)−1* 4n81p0n08a 3n50p0n18c 3n40p0n24c 3n31p0n15c 1n70p0n03b

* Where mean values for the groups differ significantly (P 0n05), the values carry different superscript letters.

Salmonella and cytokine expression (a) 25

IFN-c

IL-6

20 15 10

40 –Ct

5

Cytokine levels by real-time quantitative RT-PCR

0 25

IL-2

IL-1b

20 15 10 5 0

S.t.

(b)

E.c. S.g. S.d. S.e. CKC

IFN-c

10

S.t. E.c. S.g. S.d. S.e. CKC

IL-6

8 6 4 2 0 mRNA (fold change)

caused little damage to the cell monolayer, with any damage being most pronounced following invasion with S. enteritidis. There was only a slight increase in bacterial invasion with increased contact time of 4 h (data not shown) with no observable increase in cell monolayer damage.

–2 –4 –6 10

IL-2

IL-1β

8 6 4 2 0 –2 –4 –6 S.t.

E.c. S.g. S.d. S.e. CKC

S.t. E.c. S.g. S.d. S.e. CKC

.................................................................................................................................................

Fig. 1. (a) Quantification of cytokine mRNA in RNA extracted from uninfected CKC, or from CKC infected with Salmonella spp. or E. coli for 4 h. Cycle threshold values are expressed subtracted from 40 (the negative end point). Therefore, higher values represent higher levels of cytokine mRNA. White bars represent cytokine mRNA specific Ct values before standardization for input RNA. Black bars represent standardized values for cytokine mRNA corrected for variation in input RNA measured by 28S rRNA levels. (b) The above corrected data from (a) expressed as fold change in cytokine mRNA levels, when compared to those from uninfected CKC. Cytokine mRNA levels in uninfected CKC are set as 1. *** l P 0n01, compared to levels in E. coli-infected CKC. S.t., S. typhimurium ; E.c., E. coli ; S.g., S. gallinarum ; S.d., S. dublin ; S.e., S. enteritidis. Error bars show SEM for triplicate samples from three separate experiments.

Replicate measurements on different days were highly repeatable, with a coefficient of variation for six replicate RT-PCRs of log serially diluted RNA for the "! in Table 2. There was a different reactions as shown linear relationship between the amount of input RNA and the Ct values for the various reactions, as shown in Table 2. Regression analyses of the Ct values generated by the log dilution series gave R# values for all reactions "! 0n97 (see Table 2 for details). The increase in in excess of cycles per log decrease in input RNA for each specific "! reaction, as calculated from the slope of the respective regression line, is given in Table 2. Attempts were made to set up multiplex RT-PCRs, in which a cytokine-specific and 28S rRNA-specific reaction were carried out on the same sample in the same tube. However, there were significant differences in the cytokine-specific Ct values from the multiplex RT-PCRs compared to the single RT-PCRs. Also, the gradients generated from the regression analyses of the log "! dilution series following multiplex and single RT-PCRs were quite different. Multiplex RT-PCR therefore seemed to significantly affect the quantification of cytokine RNA. It was therefore decided to continue with single RT-PCRs, but to run both cytokine-specific and 28S rRNA-specific reactions on the same samples, in triplicate, in the same experiment, with replicate experiments. To control for variation in sampling and RNA preparation, the Ct values for cytokine-specific product for each sample were standardized using the Ct value of 28S rRNA product for the same sample from the reaction run simultaneously. The Ct values for 28S rRNA did not alter significantly from sample to sample ; the mean 28S rRNA Ct values for uninfected or infected CKC ranged from 8n58 to 8n94. Cytokine-specific Ct values varied from sample to sample, and from cytokine to cytokine. The Ct values for 28S rRNA thus appeared to be independent of cytokine production and infection. They were therefore taken to be representative of the level of RNA extracted from the CKC cultures. To normalize RNA levels between samples within an experiment, the mean Ct value for 28S rRNA-specific product was calculated by pooling values from all samples in that experiment. Tube to tube variations in 28S rRNA Ct values about the experimental mean were calculated. The slope of the 28S rRNA log dilution series "! regression line was used to calculate differences in input total RNA. Using the slopes of the respective cytokine log dilution series regression lines, the difference in "! total RNA, as represented by the 28S rRNA, was input then used to adjust cytokine-specific Ct values. Fig. 1(a) 3221

P. K A I S E R a n d O T H E R S

NO–2 [lM (5× 104 cells)–1 (48 h)–1]

8

6

4

2

0

S.t.

E.c.

S.g.

S.d.

S.e.

CKC

IFN-c

.................................................................................................................................................

Fig. 2. IFN-γ-like activity in the CM of infected or uninfected CKC. Data shown as calculated from the LPS standard curve run in each assay. HD11 cells were cultured in the presence of sample only (CM or recombinant chicken IFN-γ ; white bars), or sample which had been pre-treated with an anti-chicken neutralizing mAb (1E12, 1 : 100 dilution ; hatched bars), or the LPS inhibitor polymyxin B (10 µg ml−1 ; black bars). All assays were carried out in triplicate. Error bars show SEM. Abbreviations are as in Fig. 1.

Table 4. IL-6-like activity in the CM of infected or uninfected CKC .................................................................................................................................................

Dilution units were calculated from the dilution curve of each sample, with one dilution unit corresponding to the dilution of serum required to bring about half maximal proliferation of 7TD1 cells. Half maximal proliferation was determined from the recombinant murine IL-6 standard curve run in each assay. Values represent the mean of triplicate samples from a representative experiment. All experiments were performed at least three times. Bacterial strain

S. typhimurium F98 S. gallinarum 9 S. dublin 2229 S. enteritidis 125589 E. coli K-12 Uninfected

Dilution unitspSEM of IL-6-like activity* 77n18p3n32a 9n02p0n48b 77n37p3n90a 91n14p4n40a 20n82p0n65c 17n31p0n53d

* Where mean values for the groups differ significantly (P 0n01), the values carry different superscript letters.

shows the effect of standardizing cytokine-specific Ct values to correct for tube to tube variation in RNA levels. Standardization does not dramatically alter the distribution of the results as a whole. Fig. 1(b) shows the standardized data expressed as fold changes in mRNA levels in samples from infected CKC compared to those from uninfected CKC, which was set at a basal level of 3222

1. For statistical comparisons, cytokine mRNA changes following Salmonella infection were compared to those following E. coli infection. In terms of levels of IFN-γ and IL-2 mRNA expression, there was very little effect following infection, except that, in the case of S. enteritidis, there was an approximately fivefold decrease in IL-2 mRNA (P 0n01). For IL-1β mRNA expression, infection of CKC with E. coli caused a reduction in IL-1β mRNA levels compared to uninfected CKC. Infection with S. gallinarum or S. enteritidis had similar effects to the E. coli infection. However, compared to the E. coli infection, infection with S. typhimurium or S. dublin gave significant levels of IL-1β mRNA expression (P 0n01), similar to the uninfected controls. The most striking results were seen with regard to IL-6 mRNA expression levels. Infection of CKC with S. typhimurium, S. dublin or S. enteritidis caused a seven- to tenfold increase in IL-6 mRNA expression (P 0n01). However, following S. gallinarum infection there was a threefold decrease in IL-6 mRNA expression (P 0n01). Cytokine levels by bioassay

There are few well-characterized, reproducible, specific bioassays for avian cytokines. Bioassays to reliably measure IL-1β and IL-2 content of samples were not available for use. However, there are reliable bioassays to measure IFN-γ-like and IL-6-like activity in the chicken. CM from all CKC cultures were harvested after 4 h culture and assayed for IFN-γ-like activity using the macrophage activation factor assay. Fig. 2 shows the amount of nitrite, expressed as µM NO− (5i10% cells)−" # in the pres(48 h)−", produced by macrophages grown ence of CM from uninfected CKC, and CKC infected either with one of the four species of Salmonella, or with E. coli. As a positive control, macrophages were also grown in the presence of a 1 : 1000 dilution of recombinant chicken IFN-γ. Only very low (background) levels of nitrite were produced by HD11 cells cultured in the presence of CM from uninfected CKC, regardless of the dilution of the CM. Nitrite was produced by HD11 cells grown in the presence of CM from all the infected CKC. The quantity of NO− produced titred out with increasing dilution of the #CM down to background levels. CM from CKC infected with S. typhimurium contained most NO−, followed by that from S. # with that from S. gallinarumenteritidis-infected CKC, infected CKC containing least NO−. CM from CKC infected with S. dublin or E. coli gave#intermediate levels of NO−. # Treatment with the anti-IFN-γ neutralizing mAb, 1E12 (Lambrecht et al., 2000) removed some, but by no means all, of the NO−-inducing activity from the CM (Fig. 2), # most of the NO−-inducing activity of and neutralized # the recombinant chicken IFN-γ. Treatment with polymyxin B, an inhibitor of LPS, removed all significant NO−-inducing activity from the CM. However, at the # concentration used, polymyxin B may be toxic to the

Salmonella and cytokine expression

HD11 cells, as it also ablated some of the NO−-inducing activity of the recombinant chicken IFN-γ. # Levels of IL-6-like factor activity in the CM from the same CKC cultures were measured using the 7TD1 bioassay (van Snick et al., 1986). 7TD1 cells are dependent on the presence of IL-6 for growth and proliferation, and the assay can detect IL-6 levels as low as 0n1 pg ml−". This assay has been used to measure the presence of chicken IL-6-like activity in the CM from LPS-stimulated fibroblasts and HD11 cells (Lynagh, 1998), and in serum (Nakamura et al., 1998 ; Lynagh, 1998). Table 4 shows IL-6-like activity in the CM analysed by the 7TD1 assay, expressed as dilution units. CM from CKC infected with the broad host range serotypes (S. enteritidis, S. dublin or S. typhimurium) contained significantly more IL-6-like activity (P 0n01), than CM from CKC infected with E. coli. CM from S. gallinarum-infected CKC contained least IL-6like activity, significantly less (P 0n01) than CM from CKC infected with E. coli. DISCUSSION

IFN-γ neutralizing antibody (Lambrecht et al., 2000), but was removed by pre-treatment with the LPS inhibitor polymyxin B (see Fig. 2). The most likely explanation for these results is contamination of the CM from infected CKC with bacterial LPS (LPS stimulates HD11 cells to produce NO−), especially as the IFN-γ# like activity in the CM of uninfected CKC was very low, as were the IFN-γ mRNA levels in the uninfected CKC themselves. Invasion of S. typhimurium and S. enteritidis into avian cells produces an inflammatory acute phase response

Infection with S. typhimurium, S. dublin and S. enteritidis gave both high levels of IL-6-like activity in the CM and induced high levels of IL-6 mRNA expression in the CKC. E. coli infection gave slightly higher levels of both IL-6 mRNA and IL-6-like activity, compared to those from uninfected CKC. Finally, S. gallinarum infection seems to result in down-regulation, or non-induction, of IL-6 expression. Both IL-6 mRNA levels, and levels of IL-6-like activity, were lower in CKC infected with S. gallinarum than in uninfected CKC. There is good correlation between IL-6 mRNA levels in the CKC and IL-6-like activity in the CM from the CKC, following infection.

Although the role of cytokines in the immune response to Salmonella infection\invasion and their role in pathogenesis have been well characterized in mammals, it is poorly understood in the chicken. S. enteritidisimmune lymphokines have been partially characterized and shown to influence an inflammatory response and to activate heterophils (Kogut et al., 1994a, b, 1995). However, until the recent progress in cloning avian cytokine genes, it had not been possible to fully characterize these lymphokines due to the lack of avian cytokine reagents. It is now possible to specifically quantify, amongst others, the avian orthologues of the Th1 cytokines IFN-γ and IL-2, and the pro-inflammatory cytokines IL-1β and IL-6. The real-time quantitative RT-PCR results suggest that, with the exception of S. typhimurium, in vitro infection of CKC with Salmonella or E. coli results in the down-regulation of expression of mRNA for the pro-inflammatory cytokine IL-1β. In vivo, such down-regulation might lead to a reduced rapid inflammatory response in the gut and allow initial entry of the bacteria into epithelial cells. Infection seems to have little effect on IL-2 production, with the exception of S. enteritidis, which down-regulates IL-2 mRNA expression. With the lack of reliable IL-1β or IL2 bioassays, these levels of mRNA expression cannot be correlated with the presence of cytokine in the CM, or actual biological activity.

It would appear that production of IL-6 is not simply due to invasion. Higher levels of IL-6 were produced by CKC incubated with the non-invasive E. coli K-12. It appears that the down-regulation of IL-6 is a specific effect of S. gallinarum. IL-6 is a multifunctional cytokine that has pro-inflammatory activity via the induction of acute phase protein synthesis, and is important in the development of adaptive immune responses leading to the differentiation of B lymphocytes, cytotoxic T cells and the growth of T cells (Hirana, 1994). The role of IL6 in the pathogenesis of S. typhi and S. typhimurium has been investigated in human and murine epithelial cell lines (Weinstein et al., 1998). The invasion of S. typhi into human or murine epithelial cells results in the production of high levels of IL-6. In contrast, invasion of S. typhimurium into mice and humans results in only low levels of IL-6 production. The data presented here indicate that the invasion of S. gallinarum into nonphagocytic chicken cells results in a low level of IL-6 production, and suggests that early pathogenesis of fowl typhoid, at least in terms of IL-6 production, may more closely resemble the typhoid-like disease found in S. typhimurium-infected mice than human typhoid fever.

By contrast, assays which measure IFN-γ-like and IL-6like activity in the chicken are available, allowing quantification of IFN-γ- and IL-6-like activities in the CM from Salmonella- and E. coli-infected CKC. The real-time quantitative RT-PCR results showed little or no induction of IFN-γ mRNA expression in infected CKC compared with uninfected controls. However, the bioassay showed significant IFN-γ-like activity in the CM from all infected CKC. This activity was not removed by pre-treating the CM with an anti-chicken

The high levels of IL-6 production following invasion by S. typhimurium, S. enteritidis and S. dublin also suggest differences in the early interactions and pathogenesis of these broad host range serotypes compared with the chicken-specific serotype S. gallinarum. S. typhimurium does not frequently cause clinical disease except in very young chicks (Barrow et al., 1987), where it has been shown to cause damage to the intestinal mucosa, particularly flattening of the microvilli structure (Nagaraja et al., 1991). Although older birds rarely 3223

P. K A I S E R a n d O T H E R S

show clinical disease following S. typhimurium infection, 1-d-old birds infected orally with S. typhimurium develop intestinal lesions and flattened microvilli 2–3 d following infection (Henderson et al., 1999). Increased numbers of heterophils and mononuclear cells in the gut accompany the intestinal damage, suggesting that the mechanisms of disease resemble those found in mammals (Darwin & Miller, 1999). In contrast, S. gallinarum does not cause intestinal damage in the early stages of infection but causes an acute and virulent systemic disease with lesions of the spleen, liver and heart often accompanied by bacteraemia (Smith, 1956 ; Pomeroy & Nagaraja, 1991). The differences in IL-6 production found in this study indicate that invasion by S. typhimurium, S. enteritidis or S. dublin induces a strong acute phase inflammatory response and activation of innate and adaptive immune responses. This may contribute to both the damage of the intestinal epithelium observed following in vivo infection (Henderson et al., 1999) and also to an effective immune response that prevents systemic disease. The increased levels of heterophils found following S. typhimurium infection are also likely to play a major role in disease pathogenesis and immune protection. Heterophils have been demonstrated to play an important role in protection against S. enteritidis infection in the chicken (Kogut et al., 1994c). In mammals it has been shown that IL-8 is produced in response to Salmonella invasion (Eckman et al., 1993), along with a pathogen-elicited epithelial chemoattractant (PEEC) (McCormick et al., 1998). These act to induce an influx of PMNs that are involved in the pathogenesis of Salmonella gastroenteritis (Darwin & Miller, 1999). However, no equivalent of PEEC has yet been found in the chicken, and there are as yet no specific assays to measure IL-8 in the chicken. However, it seems likely that invasion by S. typhimurium or S. enteritidis may induce IL-8 production, and hence heterophil influx in the chicken gut. Invasion by S. gallinarum does not induce an inflammatory response

In contrast, invasion by S. gallinarum results in little or no production of IL-6. This suggests that the entry of S. gallinarum does not trigger a strong immune or inflammatory response. Such a mechanism would allow entry without intestinal damage, and may fail to trigger an effective host response allowing the development of systemic disease. The closely related S. pullorum appears to enter through chicken epithelium without causing an inflammatory response, intestinal damage or heterophil infiltration, though the main route of entry appears to be through lymphoid tissue (Henderson et al., 1999). In addition, in a human cell-culture system S. pullorum is capable of both attachment and entry into epithelial cells, but does not induce transepithelial migration of PMNs (McCormick et al., 1995). Depletion of PMNs in chickens results in systemic septicaemia following S. enteritidis infection (Kogut et al., 1994c), resulting in disease akin to that caused by S. gallinarum. It appears that inflammatory responses and PMNs in the gut are 3224

important in protection against the development of systemic typhoid-like disease, though they may result in tissue damage in birds or gastroenteritis in mammals. The failure of S. gallinarum to induce an inflammatory response in invasion in the chicken may be an adaptation that contributes to its host specific nature. However, it is likely to be of secondary importance compared to the interaction with the host’s reticuloendothelial system, which has been shown to be of prime importance in host specificity in vivo in both the chicken and mouse (Barrow et al., 1994). It is interesting that there is a down-regulation of IL-1β during Salmonella invasion. Little is known regarding the production of IL-1β by non-phagocytic cells invaded by Salmonella. IL-1β is a potent pro-inflammatory cytokine and high levels were produced in the early stages of invasion, which may act to inhibit Salmonella crossing the intestinal epithelium. This is in contrast to the production of IL-8 and PMN influx triggered by Salmonella invasion leading to damage of the gut mucosa, allowing bacterial entry (Gala! n & Sansonetti, 1996 ; Darwin & Miller, 1999). In the mouse, IL-1β is produced by macrophages following invasion across the gut epithelium and contributes to both the subsequent inflammation of the gut and pyrexia during Salmonella infection (Gala! n & Sansonetti, 1996). IL-1β is also produced by murine macrophages and dendritic cells when invaded by Salmonella in vitro (Marriott et al., 1999). The production of IL-1β by chicken macrophages in response to Salmonella is as yet undefined. In this work we have demonstrated methods for investigating cytokine production by cultured chicken cells in response to challenge with bacterial pathogens, and, though limited by the current availability of assays in the chicken, have shown differential production of cytokines in response to host specific and broad host range Salmonella. These differences suggest that pathogenesis and host specificity of S. gallinarum infection in the chicken may be related to some extent to the lack of inflammatory response in the early stages of infection in the gut. Production of pro-inflammatory cytokines, including IL-6 and probably IL-8, may limit serotypes such as S. typhimurium and S. enteritidis to the gut by induction of a strong immune response, but as a result produce lesions and flatten intestinal microvilli, the same mechanism that produces gastroenteritis in mammals (Darwin & Miller, 1999). It appears that all serotypes down-regulate IL-1β production in chicken cells, possibly to facilitate entry in vivo by inhibiting inflammation of the gut epithelium during invasion. It is anticipated that the quantitative RT-PCR techniques will be utilized to determine cytokine production from cells and tissues obtained from in vivo Salmonella infections of poultry. The interaction of Salmonella with eukaryotic cells is largely dependent on Type III secretion systems (Darwin & Miller, 1999), and their secreted proteins. Although Type III secretion systems are conserved in different bacteria [for example, the inv\spa SPI1 system is present and conserved in all Salmonella serotypes tested (Ochman & Groisman,

Salmonella and cytokine expression

1996)], it is becoming more apparent that even closely related pathogens may have a different repertoire of secreted protein effectors (Hardt & Gala! n, 1997 ; Mirold et al., 1999). It is tempting to speculate that this may play a role in the differences in the early pathogenic behaviour of different Salmonella serotypes. Further work with the host-specific S. gallinarum and broad host range salmonellae in the chicken will concentrate on studies of this matter. In addition, the application of the techniques described here will help investigation of pathological mechanisms of avian salmonellosis and other diseases at a molecular level in the chicken. ACKNOWLEDGEMENTS Our thanks go to Adrian Moody for advice on TaqMan experimental design and Scott Hulme for technical assistance. This research was undertaken with the financial support of the Biotechnology and Biological Sciences Research Council and the Ministry of Agriculture, Fisheries and Food.

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Received 2 March 2000 ; revised 3 July 2000 ; accepted 22 August 2000.